Abstract
Pseudomonas fluorescens Pf0-1 is one of the model organisms for biofilm research. Our previous transposon mutagenesis study suggested a requirement for the de novo purine nucleotide biosynthesis pathway for biofilm formation by this organism. This study was performed to verify that observation and investigate the basis for the defects in biofilm formation shown by purine biosynthesis mutants. Constructing deletion mutations in 8 genes in this pathway, we found that they all showed reductions in biofilm formation that could be partly or completely restored by nucleotide supplementation or genetic complementation. We demonstrated that, despite a reduction in biofilm formation, more viable mutant cells were recovered from the surface-attached population than from the planktonic phase under conditions of purine deprivation. Analyses using scanning electron microscopy revealed that the surface-attached mutant cells were 25 ∼ 30% shorter in length than WT, which partly explains the reduced biomass in the mutant biofilms. The laser diffraction particle analyses confirmed this finding, and further indicated that the WT biofilm cells were smaller than their planktonic counterparts. The defects in biofilm formation and reductions in cell size shown by the mutants were fully recovered upon adenine or hypoxanthine supplementation, indicating that the purine shortages caused reductions in cell size. Our results are consistent with surface attachment serving as a survival strategy during nutrient deprivation, and indicate that changes in the cell size may be a natural response of P. fluorescens to growth on a surface. Finally, cell sizes in WT biofilms became slightly smaller in the presence of exogenous adenine than in its absence. Our findings suggest that purine nucleotides or related metabolites may influence the regulation of cell size in this bacterium.
Highlights
ATP and GTP are the purine nucleotide triphosphates that are essential to drive many cellular processes in all living organisms
Our previous transposon mutagenesis study indicated that the genes involved in the de novo purine nucleotide biosynthesis are required for normal biofilm formation by P. fluorescens Pf0-1 (Newell et al, 2011b)
We sought to determine the basis for the defects in the biofilm formation by the purine auxotrophic mutants of P. fluorescens Pf0-1, in which one of the genes involved in the de novo purine biosynthesis pathway to inosine monophosphate (IMP) was disrupted
Summary
ATP and GTP are the purine nucleotide triphosphates that are essential to drive many cellular processes in all living organisms. AMP and GMP are the dephosphorylated forms of the above nucleotides and synthesized either in a de novo synthesis pathway or in a salvage pathway (Neuhard and Nygaard, 1987). In the de novo purine biosynthesis pathway, inosine monophosphate (IMP) is sequentially synthesized from 5-phosphoribosyl- -diphospahte (PRPP) in 11 enzymatic steps, where some reactions require ATP to proceed. AMP and GMP are synthesized separately from IMP as the common intermediate. The biosynthesis of purine nucleotides has a high energy cost. For this reason, another biosynthesis pathway, salvage pathway, is designed to scavenge and recycle the purine bases arising from nucleic acid turnover; adenine, guanine, and hypoxanthine are converted into AMP, GMP and IMP, respectively, by phosphoribosyltransferases (Neuhard and Nygaard, 1987). The fact that most bacteria possess both de novo purine biosynthesis pathway and salvage pathway indicates vital role of this pathway in bacteria
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
